The fifth of men, who present with high risk, localized prostate cancer (PC) account for the majority of PC- related mortality. The goals of this project are to leverage existing but untested agents and to develop new agents for imaging PC, with a focus on aggressive, localized disease. Identifying the most aggressive elements of a lesion within the prostate is critical for determining which patients should remain under active surveillance vs. those who should undergo focal therapy or prostatectomy. We propose that that can best be done through imaging, which, unlike biopsy-derived Gleason score, is not subject to sampling error. However, accurate patient selection can be accomplished only if the agents used have high specificity for aggressive PC. Mechanisms that link the prostate-specific membrane antigen (PSMA) to aggressive disease - an attribute repeatedly demonstrated pre-clinically and in epidemiologic studies - are only beginning to be uncovered, such as the relationship between PSMA expression and that of the TMPRSS2-ERG fusion, or levels of VEGF or proteolytic activity. Although a mature target for imaging and treating PC pre-clinically, PSMA gains increasing importance as one of few actionable, alternative targets to androgen receptor signaling and is increasingly pursued for treating PC and other cancers through antibody-drug conjugates, radiotherapeutics and immunotherapy. Although we reported the first low-molecular-weight imaging agent for PSMA over a decade ago, such agents are entering clinical trials only now, and remain largely untested in scenarios in which they may prove useful. We will transition to the next funding period with a pilot clinical trial using our recently descried PET agent [18F] DCFPyL to identify intra-prostatic lesions (Aim 1). As we await follow-up on those subjects, we will synthesize and test a variety of new agents with improved binding affinity, pharmacokinetics and high specificity for aggressive disease (Aim 2). A new PSMA-binding scaffold will be introduced. We will generate new hetero-bivalent compounds, with rationally chosen co-targets (along with PSMA) that historically reflect more malignant and invasive phenotypes (angiogenesis and proteolytic activity). We will provide several new targeted MR agents of high sensitivity based on multiplexing and chemical exchange saturation transfer (CEST). Introduction of MR to PSMA-targeted imaging is to leverage the clinical ubiquity of this modality and complement our radionuclide-based techniques with MR and its attendant advantages of high spatial resolution and lack of ionizing radiation (Aim 3). By focusing on imaging agents for aggressive disease we will provide an accurate picture of PC to avoid the up-staging that often obtains after prostatectomy and to opt patients out of surveillance programs only when necessary. If a positive signal is identified on PET with the agents proposed, chances are that the prostate will need to be removed - and appropriately so.